We have shown that ethanol exposure (at doses equivalent to consuming 1-2 alcoholic beverages) one day prior to prolonged ischemia and reperfusion (I/R) prevents the inflammatory responses to reperfusion after the ischemic insult on Day 2. However, the mechanisms linking events that are initiated during the period of ethanol preconditioning (ETOH-PC) on Day 1 to the reductions in postischemic microvascular dysfunction on Day 2 are unknown. We hypothesize that nitric oxide (NO) serves as an important initiating factor in ETOH-PC to trigger a cascade of signaling events that ultimately act to promote postischemic adenosine production on Day 2, thereby limiting I/R-induced leukocyte adhesion by preventing P-selectin expression. To address this postulate, we propose to determine whether: (1) NO derived from endothelial nitric oxide synthase (eNOS) initiates the protective actions of ETOH-PC; (2) ethanol-induced increases in adenosine and/or pertussis-sensitive G-protein activity provide the trigger for eNOS activation in ETOH-PC; (3) NO produced during the period of ETOH-PC initiates its protective effects by a mechanism that involves interaction of this free radical with xanthine oxidase- and/or NADPH oxidase-derived oxidants; (4) oxidant-induced, isoform-specific protein kinase C (PKC) activation contributes to the beneficial actions of ETOH-PC; and (5) formation of HO-1-catalyzed heme degradation products during reperfusion serves as the effector of ETOH-PC on Day 2 via receptor-dependent inhibition of leukocyte adhesion. To accomplish these aims, we will utilize intravital microscopic approaches to quantify oxidant production, leukocyte rolling and adhesion, and microvascular protein leakage in the small intestine of wild-type control mice (C57BL/6), in knockout mice lacking eNOS, iNOS, nNOS, xanthine oxidase, or NADPH oxidase, and in mice overexpressing distinct forms of superoxide dismutase. The influence of EtOH-PC on I/R-induced P-selectin expression will also be investigated. Isoform-specific PKC and translocation, and NOS mRNA levels and activities will be examined during ETOH-PC and I/R. The proposed studies should not only substantially improve our understanding of the mechanisms whereby ETOH-PC prevents I/R-induced microvascular dysfunction in the small intestine but should also provide a rationale for the development of pharmacologic approaches that duplicate its remarkably powerful protective effects. Moreover, this research will provide novel and important insight regarding the mechanisms whereby adenosine modulates I/R-induced P-selectin expression as well as furnish some of the first information regarding the effect of delayed ETOH-PC (or any other form of preconditioning) on platelets and their contribution to the inflammatory state induced by I/R.